Thrombin is produced by the enzymatic cleavage of two sites on prothrombin by activated Factor X (Xa).
Thrombin converts fibrinogen to an active form that assembles into fibrin. Thrombin also activates Factor XI, Factor V and Factor VIII. This positive feedback accelerates the production of thrombin.
Factor XIII is also activated by thrombin. Factor XIIIa is a transglutaminase that catalyses the formation of covalent bonds between lysine and glutamine residues in fibrin. The covalent bonds increase the stability of the fibrin clot.
In addition to its activity in the coagulation cascade, thrombin also promotes platelet activation, via activation of protease-activated receptors on the platelet.
In clinical practice the measurement of prothrombin time (PT), i.e., the time it takes for blood to clot, in terms of activated partial thromboplastin time (APTT) and activated clotting time (ACT), is widely used for screening for defects of coagulation pathways and for monitoring anticoagulant therapy.
In order to standardize the results of blood coagulation tests, the concept of International Normalized Ratio (INR) has been devised. Each manufacturer of tissue factors gives an ISI (International Sensitivity Index) for any tissue factor they make. The ISI value indicates the comparison between a particular batch of tissue factor and an internationally standardized sample. As depicted below, INR is the ratio of a patient's prothrombin time to a control sample's prothrombin time, raised to the power of the ISI value for the thromboplastin reagent used.INR=(PTtest/PTnormal)ISI 
There are two kinds of assays available for the measurement of PT. One kind of assay is coagulometric, which is based on the endpoint of clot formation from fibrinogen to fibrin conversion. The results of the assays are, however, variable, and particularly affected by interference with the fibrinogen/fibrin conversion in patients. Another kind of assay is non-coagulometric and is based on the use of synthetic substrates suitable for thrombin cleavage. The results of the latter assays are less variable and not affected by the fibrinogen level.
U.S. Pat. No. 4,061,625 discloses chromogenic thrombin substrates of the formulaD-Phe-cyclic imino acid-Arg-pNAwherein the cyclic imino acid is selected from among 2-azetidine carboxylic acid, proline, 2-piperidine carboxylic acid, and pNA is p-nitroanilide. These substrates are described as suitable for a quantitative determination of thrombin or for a study of reactions in which thrombin is formed, inhibited or consumed, or for determination of factors which exert an influence or take part in such reactions e.g., for determination of anti-thrombin, prothrombin and heparin.
When chromogenic substrates are used, the reaction is detected at approximately 405 nm. At this wavelength, whole blood can not be analysed, due to the absorption from the whole blood in its self. Chromogenic substrates are therefore not suitable when the used sample type is whole blood.
Time-resolved luminescence spectroscopy using chelates such as lanthanide chelates has for several years been applied in immunoassays and DNA hybridization assays. Due to the absorption properties of whole blood, this chelate detection technology is suitable when the sample is whole blood, compared to the chromogenic substrates, because it is possible with this technology to measure absorbance at 615 nm, at which wavelength, whole blood absorption is low.
Such chelates are described in U.S. Pat. No. 7,018,851 B1 which discloses improved fluorescent lanthanide chelates which are suitable for time-resolved fluorometric (TRF) applications. The chelates are used in specific bioaffinity based binding assays such as immunoassays.
TRF is a suitable detection technology for assays requiring high sensitivity and wide dynamic range. In immunoassay techniques using conventional fluorescence detection, high non-specific background caused by light scattering, e.g., from the biological components of the sample is a severe limitation to the sensitivity of the assay.
The fluorescent lanthanide chelates have traditionally been used in immunoassays where the detection principle is based on the capture of such chelates and detection thereof. In contrast they have not been used in enzyme assays, where the detection principle is based on cleavage of the chelate substrate and detection of the captured (not cleaved) substrate remaining in the assay.
The key feature of such a system is the provision of a substrate which is on the one hand stable and on the other hand specifically cleavable by thrombin.
Therefore, there exists a need for an enzyme substrate which is specifically cleavable by thrombin and which comprises an intrinsic stable luminescent component having a long lifetime, thus facilitating detection in whole blood and resulting in less interference.
Furthermore there exists a need for an assay which is fast, easy to perform, subject to low variability, easily automated, and of low cost.
Surprisingly it has been found that by combining, via a certain linker, a peptide which is specifically cleavable by thrombin, with a luminescent chelate, a stable substrate with a long lifetime, which can be used when the sample type is whole blood, is provided.